Retentive chromatographic separations are chromatographic methods where biomolecules in a sample are retained on a chromatography resin and are subsequently eluted. Retentive separations can use either batch adsorption techniques, i.e., where the absorption takes place in a slurry with the chromatography resin, or by column absorption techniques, i.e., where the adsorption takes place by running a solution containing the biomolecules through a column. In either case of retentive chromatographic separation, the biomolecules can be bound to the column resin, e.g., by an ionic, hydrophobic, or affinity interaction.
Following binding of the biomolecules to the chromatography resin, the biomolecules are eluted from the column. Certain methods of elution allow the skilled artisan to specifically control the point in the elution that the biomolecules of interest are released from the resin. These methods include, but are not limited to, use of a salt gradient, gradually mixing an aqueous buffer with an organic solvent, or the gradual addition of detergents. Based on the conditions used, a biomolecule may elute from a column very sharply, i.e., in a small number of fractions, or it may elute more broadly, i.e., in a larger number of fractions. It is often possible for the skilled artisan to adjust the elution conditions to optimise the separation of biomolecules of interest from unwanted material during elution, and to sharpen the peak at which a biomolecule elutes. This is referred to as improving the resolution of the chromatography process.
The standard method of assessing the resolution of a chromatographic separation is for a skilled operator to monitor the elution profile with a UV detector for absorbance at 280 mm, which provides a rough measurement of total protein eluting in a given fraction. In the case that an operator is eluting a column by means of, for example, a buffer solution of gradually changing salt gradient, if the UV detector shows an increase in the amount of protein in the eluate discharged from the column then the operator could arrange for this fraction of the eluate to start to be collected in a reservoir in a fraction collector.
The collection of the fraction would continue until the amount of protein detected in the eluate dropped below a threshold value at which point the eluate discharged from the column could be collected in a different reservoir in the fraction collector or discharged to waste. As it is very wasteful to have a trained operator continuously monitoring a chromatography run then it is usual to perform the analysis of a sample in two (or more) runs.
In the first run the sample is separated in a column while using a user chosen gradient which it is hoped will produce a satisfactory separation. The resulting chromatogram is then analysed be the operator who then defines a new, optimised gradient which is expected to give a better separation. The optimised gradient may have a different starting point and/or end point and/or slope and/or number of steps. The purpose of the optimisation is to modify the separation of the sample so that the peak(s) containing the biomolecule(s) of interest is(are) far away from the peaks containing contaminants. The sample is then separated in a second, optimised run using the newly defined gradient. If the separation is still not satisfactory then the user may redefine the optimised gradient and perform a further run or runs.
An automated system for performing a separation using a gradient is described in U.S. Pat. No. 5,112,949. A problem with such manual and automated systems with gradually changing gradients is that if the gradient is made shallow then it takes a lot of time to perform the elution and if the gradient is made steep then instead of each biomolecule of interest being eluted in turn the elution of the biomolecules overlap. This leads to several species of biomolecules being collected in each fraction instead of each specie of biomolecule being collected in its own, separate fraction.